The invention relates to a new tool for transfer in eukaryotic cells or a tool for production in the bacterial culture supernatant of chimeric proteins from the type III secretion system present in the Pseudomonas bacteria, especially Pseudomonas aeruginosa. More precisely, the invention relates to new chimeric protein expression vectors, or more generally amino acid sequences in P. aeruginosa. It also relates to P. aeruginosa strains transformed with the said expression vectors to secrete or inject using TTSS in P. aeruginosa, chimeric proteins in the culture supernatant or in the eukaryotic cells respectively. Thus an in vitro or ex vivo injection method (also called translocation) of chimeric proteins in eukaryotic cells, as well as a chimeric protein production method in the culture supernatant, methods that can be used for therapeutic purposes also form an object of this invention.
Bacteria naturally possess protein secretion systems enabling them to send enzymes or toxins at distance. In addition to these systems, some of them also possess a “type III” secretion system (called TTSS) that allow them to directly inject toxic proteins from their cytosol into the cytosol of a target cell. This secretion mechanism is complex and comprises a certain number of elements that form a canal cutting across the inner membrane, periplasmic space and the outer membrane of the bacteria. This canal allows the transfer of specific toxins. In most species of bacteria, this secretion system can be activated either by contact with a target eukaryotic cell, that triggers the injection of toxins into the cytosol of the target cell, or by Ca2+ chelation in the outer medium, that triggers the secretion of specific toxins in the culture medium.
It is thus interesting to study whether these type III secretion systems (TTSS) can be used to inject proteins of interest into eukaryotic cells by fusing the nucleotide sequence encoding the toxin secreted by the TTSS with a second nucleotide sequence encoding a protein of interest.
A TTSS was described in the Yersinia bacteria by Cornelis et al. (Molecular Microbiology 23(5), 861-867 1997). This document indicates that the type III secretion system of Yersinia enterocolitica, called Ysc, is capable of secreting and then translocating effector proteins mainly YopE and YopH in the host cell. Sory et al. (Proc. Natl. Acad. Sci. USA 92, 11998-12002 1995) studied the molecular structure of Yop proteins and highlighted the presence of three domains in YopE and YopH, a N-terminal domain for secretion, a domain for translocation and a C-terminal region responsible for the toxic activity of the said protein. These properties were highlighted by constructing hybrid proteins obtained by fusing the amino-terminal part of YopE or YopH with a reporting enzyme, such as adenylcyclase activated by calmodulin, the enzyme activity being measured subsequently.
Moreover, U.S. Pat. No. 5,965,381 indicates the advantage of precisely identifying the minimum size of the nucleotide sequence encoding the effector protein required for secretion and subsequent transfer of a target protein into an eukaryotic cell. The method described to perform this is similar to the previously mentioned method that involves fusing sequences containing a decreasing number of nucleotides with a sequence encoding a reporting enzyme activated by calmodulin, the appearance of cyclase activity in the cytosol of the translocated cell showing the existence of translocation. Even if it is concluded from the description that the constructions were carried out from the first 50, 71, 100 and 130 amino acids of YopE, only the 130 amino acid fragment is illustrated, which means that the other identified amino acid sequences are too small to allow secretion and subsequent translocation of the protein of interest. Consequently, a high number of amino acids required for secretion (130 amino acids) limit, if not prohibit the possible use of this sequence for transferring large-size proteins of interest.
U.S. Pat. No. 6,306,387 describes the presence of a type III secretion system in the Salmonella bacteria. This document especially illustrates the possibility of translocating into host cells, a chimeric protein named SptP-NPc obtained by fusing the 173 amino acid N-terminals constituting the toxic protein SptP, naturally secreted by the type III system of Salmonella with a partial sequence encoding an Influenza virus nucleoprotein, with the detection being carried out using antibodies capable of detecting the chimeric protein in the infected cell. Even in this case, the amino acid sequence of the injected toxic protein is relatively long as it comprises as much as at least 173 amino acids, thus prohibiting the transfer of large fused proteins into the host cell. Since the injected proteins were not cleaved at a later stage, it is essential to use a toxic protein having the smallest possible size.
The existence of a type III secretion system was also illustrated in a third genus of bacteria, Pseudomonas, especially of type Pseudomonas aeruginosa. P. aeruginosa is a Gram-negative bacillus, and an opportunistic pathogen responsible for serious nosocomial infections in hospitals. It is also one of the main causes of morbidity and mortality in people suffering from mucoviscidosis. The type III secretion system of P. aeruginosa is a very effective defence especially against phagocytes, mainly neutrophil granulocytes, the first line defence in cellular immunity. This system is also used by the bacteria to trigger the proliferation and apoptosis of T lymphocytes. It is known that P. aeruginosa secretes four toxins, namely ExoS, ExoT, ExoY and ExoU using the type III secretion system.
ExoS is a protein comprising approximately 454 amino acids; sequences for ExoS are known and published in the NCBI GenBank, for example, accession nos. NP252530, AAK39624, AAK38480, AAK38452, AAK38479, AAK38436, AAK34869, AAK38419, AAK38477, AAK38421, AAK37418, AAK38437, AAK38420, AAK38478, AAK38417, CAA67834, AAG07228, and AAA66491. Ahead of the gene encoding ExoS, there is a nucleotide sequence that may encode a molecular chaperone protein supposedly specific to ExoS, named “Orf 1”. ExoS possess an ADP-ribosylation activity on small Ras family G-proteins, as well as the capacity to activate the Rho-GTPases. ExoT possess an ADP-ribosylation activity as well but is 100 times inferior to ExoS. Moreover, it is known that 75% of the nucleotide sequences of ExoT and ExoS are identical (according to the LFASTA programme, W. R. Pearson & D. J. Lipman PNAS (1998) 85:2444-2448). The mode of action of ExoU is unknown but involves a powerful cytotoxin on the epithelium and macrophages.
Polack et al. (Biochemical and Biophysical Research Communications 275, 854-858, 2000) refers to a natural isolated strain of P. aeruginosa, whose characteristics are not mentioned (CHA strain). The strain is transformed with a pBP31 plastid mainly containing two fused nucleotide sequences, corresponding respectively to the nucleotide sequence encoding the 129 amino acid N-terminals of ExoS and the sequence encoding the p67phox protein involved in reconstituting the NADPH oxidase enzyme complex. Polack showed the capacity of this transformed strain to not only secrete the p67 protein but also to reconstitute the NADPH oxidase activity, following injection of the fused protein into the cytosol of EBV-B lymphocytes, thus illustrating the intracellular activity of the injected proteins. The 129 AA sequence of ExoS used by Polack is thus the smallest sequence ever described for injecting target proteins.